The nef gene of the type 1 human immunodeficiency virus (HIV-1) has remained the most enigmatic and controversial of the nine genes comprising this pathogenic human lentivirus. Early studies suggested that nef functioned as a negative factor by suppressing viral transcription mediated through the HIV-1 long terminal repeat thereby producing an inhibition of viral replication. Accordingly, it was proposed that Nef might play a central role in the establishment or maintenance of viral latency. In contrast, more recent studies, in part supported by this grant, have challenged these results leading instead to the conclusion that nef functions as a positive rather negative factor in the HIV life cycle. Specifically, these investigations have shown that Nef significantly enhances the intrinsic infectivity of HIV acting after virus binding and cellular entry but prior to proviral gene transcription. In addition to these enhancing effects on viral infectivity, Nef promotes the internalization and subsequent degradation of surface CD4, the major high affinity receptor for HIV-1. While this response likely serves to limit virus superinfection, additional functional effects may occur including alterations in the state of T cell activation. Increased intracellular activation may in turn facilitate the completion of HIV reverse transcription, nuclear import, and/or integration within the resting cell. At this juncture, it remains unknown whether these two different functions of Nef, enhancement of viral infectivity and down regulation of CD4, are mechanistically linked or unlinked processes. These studies of nef have taken on even greater significance with the discovery in the simian immunodeficiency virus system that the nef gene is required both for the production of high viral burdens in vivo and the clinical appearance of simian AIDS. These new insights into the growth promoting and pathogenic effects of Nef underscore the potential therapeutic value of effective Nef antagonists. However, the development of such Nef antagonists will almost certainly require a more complete understanding of how the Nef protein exerts its biological effects including its intracellular site of action, the structure-function relationships that control this activity, and the nature of the cellular cofactors that interact with Nef and contribute to its function. In the present proposal, an experimental plan has been developed that directly addresses all of these important questions. The Specific Aims of the present grant request include: 1) to determine the biochemical basis for HIV Nef enhancement of viral infectivity, 2) to identify structural domains within HIV Nef that are required for the enhancement of viral infectivity and/or down regulation of CD4, 3) to identify cellular proteins that physically interact with HIV Nef and to delineate their role in the biological functions of Nef, and 4) to prepare human T cell lines conditionally expressing Nef to permit dynamic studies of CD4 receptor down regulation and analysis of potential effects of Nef on the intrinsic state of T cell activation. The insights derived from these experimental studies should lead to a fuller understanding of the mechanism HIV nef gene function and hopefully to new approaches to inhibiting HIV replication and/or preventing disease progression.